Regulation of Stem Cell Self-renewal and Differentiation NIH 1 R01 GM080501 Adult stem cells are required throughout life to replenish differentiated cells and repair damaged tissue. The mechanisms that regulate self-renewal vs onset of differentiation in adult stem cell lineages are key for regenerative medicine as well as understanding the genesis and biology of cancer. We propose to investigate how interactions with somatic support cell partners regulate the decision between self renewal and differentiation in the Drosophila male germ line stem cell lineage, a powerful system for study of adult stem cells in vivo in the context of signals from their local environment. In previous funding cycles, w discovered that somatic cyst cells in the testis, functionally equivalent to mammalian Sertoli cells, provide a crucial microenvironment that regulates the switch from germ line stem cell to transit amplifying spermatogonia. We now propose to utilize the powerful system and tools we have established to investigate the molecular circuitry that regulates this key state transition in the male germ line stem cell lineage in response to cues from somatic support cells. To discover the intrinsic factors that maintain early germ cells in a state to either self-renew or initiate differentiation in response to their microenvironment, we will test models for transcriptional regulatory circuitry suggested from bioinformatic analysis of genes coordinately upregulated as germ line stem cells become TA spermatogonia, including the possible roles of the bHLH transcription factor Daughterless and its inhibitor Emc, and the Zn finger transcription factor Klu a regulator of stem cell state in neuroblast lineages, the repressor Aef-1, and the activator rotund. To elucidate whether and how cyst cells send a go differentiate signal to instruct germ line stem cells to exit self-renewal and enter the spermatogonial program of limited transit amplifying (TA) divisions, we will identify and investigate the role of genes encoding predicted secreted or cell surface proteins that are required in somatic cyst cells for early germ cells to properly initiate differentiation as transit amplifying spermatogonia, starting with Gp150, a regulator of Notch pathway signaling, identified in the initial rounds of our cell type specific RNi screen. Our results may elucidate an alternative view of the stem cell niche, in which differentiation signals play key roles, and will illuminate how close-range reciprocal cell-cell signaling can coordinate co-differentiation of disparate cell types that must work together to make functional tissues.